1. Field of the Invention
The invention herein relates to the extraction of natural products from microorganisms, especially algae. More particularly it relates to control of large size aqueous photosynthetic bioreactor systems to obtain such products from the microbial genus Haematococcus, which has heretofore only been cultured in laboratory environments in small containers.
2. Description of the Prior Art
In view of environmental constraints, economics, and various other factors, astaxanthin, which is currently produced by chemical synthesis from petroleum- or coal-derived raw materials is coming into disfavor or is facing restrictions or outright bans from the marketplace. For instance, aniline-based sources of astaxanthin dyes are or will soon be phased out by government regulation in Europe, and similar restrictions or bans are likely to arise soon in other areas of the world, including the United States. Consequently there is a major research and development effort going on worldwide to find natural sources for astaxanthin.
It has been known that astaxanthin can be obtained from certain species of microorganisms, in particular those of the unicellular alga genus Haematococcus. Upon exposure to light, this alga genus produces astaxanthin by photosynthesis. If Haematococcus species, such as H. pluvialis, could be cultivated on a mass production basis, a valuable and economic natural source for this dye would be available.
The obvious utility of mass production of photosynthetic microorganisms resides in the process of photosynthesis itself. Given the appropriate supply of light, water, carbon dioxide (CO.sub.2), and control of temperature, pH and the aqueous environment motions (e.g., turbulence), photosynthetic microorganisms can utilize sources of essential nutrients such as nitrogen (N) and phosphorous (P) to convert solar energy into chemical energy. Thus, the process of growing or culturing photosynthetic microorganisms involves the introduction of nutritionally complete medium to a contained volume of culture, maintenance of optimal growth conditions in that volume, and subsequent harvest or removal of the microbial cells from the spent medium. All culture programs must devise methods to accomplish each of these phases of the production process efficiently.
Of these requirements the most difficult to achieve is usually the step of maintaining optimum growth which equates to economically feasible production. Haematococcus species from which astaxanthin is derived are very sensitive, by an order of magnitude, to small changes in growth environment. It is common, for instance, for a researcher to develop a protocol for maintaining H. pluvialis in the laboratory in a small volume in a laboratory container, only to have the system fail dramatically in large size volume required for economic field production, where control of system parameters is much more difficult and more variables can be encountered.
For example, one photosynthetic growth mechanism, known as the "flashing light effect," i.e., the ability of some photosynthetic cells to effectively use energy from an intermittent light source see, e.g. Emerson, et al., J. Gen Physiol., 15(4):391-420 (1932)!, has not been effectively utilized in a mass culture system, because it depends on the turbulent flow regime in the growth media that exposes cells intermittently to a light source. Too much or too little exposure results in decreased production of microorganisms. A method or apparatus has been developed by Applicants which maximizes the flashing light effect by use of controlled turbulent flow regime such that this effect could be utilized in a mass culture system, thus enhancing the efficiency and productivity of that system for Haematococcus spp.
It has been known that one must avoid excessive shear in the fluid on the scale of the microorganism, in order to prevent destruction of the microbial mass. For instance, Thomas et al., J. App. Phycology, 2:71-77 (1990) reported that in small gap rotating cylinders at Reynolds Numbers above 116 (and up to 3500) cell growth rate became negative, i.e., that the microbial mass was dying, not growing. Thus, the dimensions or mechanical configuration of the apparatus must also be controlled in order to alleviate the deleterious effects of turbulence on cell integrity.
The concept of culturing microorganisms in open or closed systems and harvesting a product from the microorganisms is not new. There are numerous systems used throughout the world in which algae or phytoplankton are grown and harvested, either for direct usage (as for animal food) or for indirect usage (as sources of chemicals such as carotenes). However, almost all such systems operate on only a very few species of microorganisms; i.e., those which have been found to be tolerant to the widest range of growth conditions and which produce relatively simple products. This in turn has limited the industry to production of a very small number of products compared with what has been extracted in laboratory scale, experimental cultures. Efforts that have been made to culture Haematococcus for production of astaxanthin have been failures because laboratory cultures have not been made to survive or grow economically in large scale volumes. Furthermore, Haematococcus spp. which will tolerate the fairly crudely controlled processes of the past (either open or closed systems) have not been capable of yielding astaxanthin, either directly or indirectly, in economical quantities. Haematococcus has not proved amenable to culture in the current systems, since such systems cannot be controlled in a sufficiently precise manner to maintain adequate health and growth of Haematococcus which may have been grown on a small scale in a laboratory. Laboratory scale apparatuses generally do not contain turbulent flows, while commercial production scale systems do. Heretofore, the necessity of control of turbulence has not been recognized as a crucial factor in mass culture apparatus, as the Applicants have found to be necessary for the health of Haematococcus microorganisms.
Numerous operating conditions or factors are critical in commercially viable production of astaxanthin from condition-sensitive Haematococcus spp. Consequently, a system in which precise control of these operating conditions is accomplished will permit not only large scale culturing of Haematococcus spp., which cannot now be grown by existing commercial technology, but also the mass production of natural astaxanthin which is not currently available on an economic basis in the marketplace.